Abstract: Gliomas, with glioblastomas (GBM) in particular, are one of the most lethal human malignancies due to lack of success with current conventional treatments due to their invasive nature and heterogeneity. We will leverage expansion microscopy, a novel super-resolution microscopy method, as a platform to develop novel tools for highly multiplexed in situ analysis to generate comprehensive maps of GBM spatial heterogeneity and structure. We will use these tools to help understand the molecular and morphological phenotypes of GBM invasion and pathology, which we hope will guide future therapeutic interventions. The proposed research will consist of three aims which seeks to develop novel in situ analysis tools driven by biological questions in GBM organization: (1) Comprehensively map cell types and states within GBM tumor tissue with high spatial resolution. To enable this, we will develop an innovative method for highly multiplexed readout of RNA in expanded tissues with tailored in situ gene expression panels based on single-cell RNA sequencing signatures. (2) Study the nanoscale interactions between tumor cells and their surrounding microenvironment that are implicated GBM invasion. To accomplish this, we will develop an approach for multiplexed protein imaging with nanoscale resolution using DNA barcoded antibody libraries. (3) Develop a scalable analysis platform with automation and analysis software to integrate multiplexed RNA and protein imaging in the same sample. We will apply this platform to patient derived tumor samples to understand the molecular and morphological phenotypes of invading GBM tumor cells, as well as build a map of the spatial heterogeneity of GBM. These novel technologies to spatially map molecular information in complex tissues will not only be invaluable for understanding glioma and cancer, they will be broadly applicable to many other spatially complex biological processes.